This invention relates to the treatment of obesity in animals. In particular, the invention relates to the treatment of obesity in humans, although it is to be understood that the present invention also extends to the treatment of obesity in non-human mammals, for example, for the improvement of meat qualities in farm animals used in food production.
The critical role of human growth hormone (hGH) in postnatal growth in humans is well recognised. Less obvious is the impact of this hormone on the regulation of lipid and carbohydrate metabolism, due to lack of detailed molecular studies.
It is well documented that the predominant form of hGH is a globular protein with a molecular weight of 22,000 daltons (22-KD) and consists of 191 amino acid residues in a single-chain, folded by 2 disulphide bonds with a small loop at the carboxyl terminus between residues 182 and 189. Recent crystallographic studies also show that the hGH molecule contains four anti-parallel xcex1-helices which are arranged in a left-twisted, tightly-packed helical bundle1. The concept that there are discrete functional domains within the hGH molecule responsible for specific metabolic actions of the hormone is generally accepted. The amino-terminus has been identified as the functional domain responsible for the insulin-like actions of the hGH molecule2,3.
Recombinant DNA technology opens the way to the large-scale commercial production of human growth hormone, and the recombinant hGH appears to have equivalent biological efficacies and pharmacokinetic properties4,5. Current supply of this multiple-functional hormone no longer restricts the types and numbers of experimental therapies in humans and animals. The use of hGH for treatment of short stature in children and adults is well-established6. Therapeutic effects of hGH in female infertility have also been reported7,8. Treatment of human obesity with hGH encounters a variety of problems. Evidence suggests that this multiple-functional hormone often simultaneously exerts in vivo, by various bioactive domains within the molecules, some adverse effects9,10.
Regulation of lipid metabolism by GH was first described in 1959 by Raben and Holienberg11. The regulatory role of the hormone in lipid metabolism was subsequently supported by the body composition studies of GH-deficient and GH-treated humans12,13 and pigs14,15. The findings of Gertner suggest that hGH is linked to adipose tissue distribution through a series of interactions known as the xe2x80x9cGH-fat cyclexe2x80x9d16. However, the molecular events transpiring to these biochemical and physiological changes remained largely unknown. The metabolic effects of GH on adipose and other tissues in vivo are variable and complex, apparently consisting of at least two components, an early insulin-like effect followed by a later more profound anti-insulin effect17. The results of the latter effect may include both a stimulation of lipolysis and an inhibition of lipogenesis. The anti-lipogenic effect of hGH has been substantiated with the demonstrations of the decrease of the expression of glucose transporter GLUT 4 in adipocytes18, the inhibition of the activity of acetyl-CoA carboxylase in adipose tissues19,20 and the reduction of glucose incorporation into lipid in both isolated cells and tissues21,22.
In view of the multiple-functional effects of intact hGH and the problems encountered in clinical applications of the intact hormone, work leading to the present invention has been directed to investigating whether hGH derivatives could be synthesised that retain the desired bioactivities and lack the unwanted side effects.
The structure-function studies of hGH with synthetic hormonal fragments have revealed that the carboxyl terminus of the hGH molecule appears to be the functional domain of the hormone for the regulation of lipid metabolism20,23 and it has been shown that a synthetic peptide having a sequence based in the carboxyl terminal region reduces body weight gain and adipose tissue mass in a laboratory obese animal model.
The entire contents of U.S. Pat. No. 5,869,452, issued on application Ser. No. 08/340389, dated Nov. 15, 1994, including the specification, claims and figures, are incorporated herein by reference in their entirety.
The present invention provides a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone. The peptide may comprise an analogue of the carboxyl-terminal sequence of human growth hormone or the growth hormone of a non-human mammalian species. As described above, the carboxyl-terminal sequence of growth hormone includes a bioactive lipid metabolic domain. In one embodiment of the invention, the peptide comprises an analogue of the carboxyl-terminal sequence of human growth hormone containing amino acid residues 177-191 or a corresponding sequence of a non-human mammalian growth hormone. The analogue may be obtained by insertion, deletion or substitution of amino acids in, or chemical modification of, the native carboxyl-terminal sequence of human growth hormone or the growth hormone of a non-human mammalian species.
In another aspect, the present invention provides a method for treating obesity comprising administering an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone, as described above. The treatment may be administered to any animal, including humans.
The present invention also provides a pharmaceutical composition for use in the treatment of obesity comprising an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, together with one or more pharmaceutically acceptable carriers and/or diluents.
In yet another aspect, the present invention provides use of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, in the manufacture of a pharmaceutical composition for the treatment of obesity in an animal.
According to one aspect of the present invention, there is provided a method for the treatment of obesity in an animal, which comprises administering to the animal an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone.
Preferably, the animal is a human although the invention also extends to the treatment of non-human mammals Preferably also, the peptide comprises an analogue of the carboxyl-terminal sequence of human growth hormone containing amino acid residues 177-191 (hereinafter referred to as hGH 177-191). Alternatively, the peptide may comprise an analogue of the carboxyl-terminal sequence of the growth hormone of other non-human mammalian species, such as bovine, porcine, ovine, equine, feline or canine growth hormone, corresponding to the hGH 177-191 peptide.
As used throughout this specification, the term xe2x80x9cobesityxe2x80x9d is used to include both excess body weight and excess adipose tissue mass in the animal, and correspondingly the references to treatment of obesity include both reduction of body weight gain and reduction of adipose tissue mass of the obese animal.
The expected outcome of any treatment of obesity is the reduction of body weight, body adipose tissue mass in particular. The reduction of body adipose tissue mass is directly regulated by two biochemical processesxe2x80x94lipogenesis (fat-production) and lipolysis (fat-reduction)xe2x80x94and it is generally understood that these biochemical processes are controlled by key metabolic enzymes, specifically the fat-reducing key enzyme (hormone-sensitive lipase) and the fat-producing key enzyme (acetyl CoA carboxylase).
It has been shown by the present inventors that hGH 177-191 is effective in stimulating the fat-reducing key enzyme, hormone-sensitive lipase, and in inhibiting the fat-producing key enzyme, acetyl CoA carboxylase. This is further supported by data showing that in the presence of hGH 177-191, fat utilization is accelerated while fat production is reduced, as measured by metabolic end-products in vitro as well as in vivo. In addition, the mechanism of these molecular actions has been established as resulting from the activation of the production of the cellular second-messenger, diacylglycerol.
It will, of course, be appreciated that the present invention extends to the use of peptides which are analogues of longer amino acid sequences than the particular sequence 177-191 of growth hormone, for example analogues of the sequence 172-191 of human growth hormone or the corresponding sequence of growth hormone of other non-human mammalian species.
The concept of correspondence in amino acid sequences between species is well known in the biological sciences and is determined by aligning comparable sequences (including if necessary theoretical deletions) to match isofunctional or isostereo amino acids thereby maximizing homology. The published corresponding sequences of the C-terminus region of the growth hormone of selected mammals are tabulated below26, using standard single letter notation: (SEQ ID NOS 34-52, respectively in order of appearance).
The present invention extends to the use of peptides which are analogues of the native carboxyl-terminal sequences of human growth hormone or growth hormone of other animal species, and which are derived from natural or synthetic (including recombinant) sources, provided always that the resulting peptide retains the biological activity of the native carboxyl-terminal sequence described herein, namely the ability to reduce body weight gain and adipose tissue mass in an obese animal. In particular, these analogues may exhibit a cyclic configuration, which may be induced by a disulfide bond.
The analogues of the present invention may be derived by elongation, insertion, deletion or substitution of amino acids in, or chemical modification of, or introduction of a cyclic amide bond between the side chains of amino acids of, the native carboxyl-terminal sequence. Amino acid insertional analogues include amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple (for example, up to 10, preferably up to 5) amino acids. Insertional amino acid sequence analogues are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional analogues are characterised by the removal of one or more (for example, up to 5, preferably up to 3) amino acids from the sequence. Substitutional amino acid analogues are those in which at least one amino acid residue in the sequence, preferably one or two, has been replaced by another of the twenty primary protein amino acids, or by a non-protein amino acid. Chemical modifications of the native carboxyl-terminal sequence include the acetylation of the amino-terminus and/or amidation of the carboxyl-terminus and/or side chain cyclisation of the native carboxyl-terminal sequence.
Analogues of the native carboxyl-terminal sequences of human growth hormone or growth hormone of other animal species which in particular retain the same conformation, structure and charge characteristics as the native carboxyl-terminal sequences can be expected to exhibit the same or similar biological activity as the native sequences, in particular in the ability to reduce body weight gain and adipose tissue mass in an obese animal.
Whilst the following detailed description refers specifically to analogues of hGH 177-191, it is to be understood that this invention extends to similar analogues of corresponding peptides of non-human mammalian growth hormone as described above.
Peptides comprising amino acid residues 177-191 of native human growth hormone (hGH 177-191) include the following sequence (Ref No. 9401):
Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe (SEQ ID NO: 1)
Such a native peptide may be in cyclic disulfide form, and may comprise an organic or inorganic acid addition salt.
Analogues of the hGH 177-191 peptide may be obtained by deletion or insertion of one or more amino acid residues at any position along the native sequence, with the retention of anti-obesity properties as described above. Preferably, the analogue is in a cyclic configuration.
Alternatively, analogues of the hGH 177-191 peptide may be obtained by substitution of one or more amino acid residues at any position along the native sequence.
Screening for in vitro and in vivo activity using alanine substitution scanning and other methods reported herein has revealed positions and relationships between amino acids in hGH 177-191 which are important in the bioactivity as described above. Preferred analogues of the current invention include peptide analogues of hGH 177-191 wherein
(i) amino acids at positions 182 and 189 of hGH are joined by a bond to promote a cyclic conformation; and/or
(ii) amino acids at positions 183 and 186 of hGH are joined by a salt bridge or a covalent bond.
The bond between amino acids at 182 and 189 of hGH may be a disulfide bond, in which case the amino acids at positions 182 and 189 of hGH may preferably be L- or D-Cys or Pen.
When the amino acids at positions 183 and 186 of hGH are joined by a salt bridge, these amino acids may preferably be (X and Y) or (Y and X), respectively, where:
X is a positively charged amino acid such as L- or D-Arg, Lys or Om and
Y is a negatively charged amino acid such as L- or D-Asp or Glu.
When the amino acids at positions 183 and 186 of hGH are joined by a covalent bond, that bond may be an amide bond in which case these amino acids may preferably be (X and Y) or (Y and X), respectively, where:
X is selected from the group consisting of L- or D-Lys and Om and
Y is selected from the group consisting of L- or D-Asp and Glu.
The amino acid at position 178 of hGH is preferably a positively charged amino acid such as L- or D-Arg, Lys or Om.
Analogues may also be obtained by elongation of the native hGH 177-191 peptide sequence at one or both ends of the amino acid residues, for example with one or more hydrophilic amino acids to increase solubility in aqueous solution. Such analogues include the following sequence, preferably in cyclic disulphide form:
X1m-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-X2n (SEQ ID NO: 2)
wherein X1 and X2 are each is selected from the group consisting of L- or D-Arg, His and Lys, and m and n are each 0, 1, 2 or 3 with the provision that at least m or n is 1.
Throughout this specification, elements which are underlined denote differences from the native hGH 177-191 sequence, and unless otherwise stated, amino acids at positions corresponding to 182 and 189 are joined by a disulfide bond.
One elongation analogue not elongated with a hydrophilic amino acid but nonetheless exhibiting especially enhanced anti-obesity properties is the following (Ref No. 9604):
Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe. (SEQ ID NO: 19)
Analogues may also be obtained by chemical modification of the native hGH 177-191 peptide sequence. Such analogues include the sequence:
Y1-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe (SEQ ID NO: 3)
wherein Y1 is selected from the group consisting of the desamino form (H), acetyl (CH3COxe2x80x94) and other acyl groups; or the sequence:
Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-Y2 (SEQ ID NO: 4)
where Y2 is selected from the group consisting of xe2x80x94CONH2 and alkyl amide groups.
Specific hGH 177-191 analogues obtained by substitution of amino acids, by elongation, by chemical modification, or by introduction of a cyclic amide bond between side chains of amino acids, of the native hGH 177-191 peptide sequence, and which exhibit anti-obesity properties, include the following:
wherein the amino acid residue abbreviations used are in accordance with the standard peptide nomenclature:
All amino acids, except for glycine, are of the L-absolute configuration, unless indicated as D-absolute configuration. All the above peptides above have a cyclic disulfide bond between Cys(182) and Cys(189) or Pen(182) and Pen(189) as a appropriate.
Where appropriate, the analogues described above may comprise an organic or inorganic acid addition salt.
The term xe2x80x9ceffective amountxe2x80x9d as used herein means an amount of the peptide sufficient to attain the desired effect in the treatment of obesity in the animal, but not so large an amount as to cause serious side effects or adverse reactions.
In another aspect, the present invention provides the use of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, in the treatment of obesity in an animal or in the manufacture of a pharmaceutical composition for the treatment of obesity in an animal.
In yet another aspect, the present invention provides a pharmaceutical composition for use in the treatment of obesity in an animal, comprising an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, together with one or more pharmaceutically acceptable carriers and/or diluents.
The peptide which is the active ingredient of the pharmaceutical composition of this aspect of the invention exhibits advantageous therapeutic activity in the treatment of obesity in an animal when administered in an amount appropriate to the particular case. For example, from about 0.5 xcexcg to about 20 mg per kilogram of body weight per day may be administered. Dosage regimens may be adjusted to provide the optimum prophylactic or therapeutic response. For example, one or more divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the clinical situation.
The active ingredient may be administered in any convenient manner such as by the oral, parenteral (including intraperitoneal, intravenous, subcutaneous, intramuscular and intramedullary injection), intranasal, intradermal or suppository routes or by implanting (eg using slow release devices). For ease of administration, oral administration is preferred, however parenteral administration is also quite convenient Depending on the route of administration, the active ingredient may be required to be coated in a material that protects said ingredient from the action of enzymes, acids and other natural conditions which may inactivate the said ingredient. For example, low lipophilicity of the ingredient may allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the composition by other than parenteral administration, the active ingredient may be coated by, or administered with, a material to prevent its inactivation.
The active ingredient may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations will usually contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomorosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by, for example, the use in the compositions of agents delaying absorption.
Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the active ingredient is suitably protected as described above, the composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.01% by weight and more preferably at least 0.1-1% by weight of active ingredient. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active ingredient in the pharmaceutical compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention may, for example, be prepared so that an oral dosage unit form contains between about 0.5 xcexcg and 200 mg and more preferably 10 xcexcg and 20 mg of active ingredient.
The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained-release preparations and formulations.
As used herein, pharmaceutically acceptable carriers and diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmnaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the treatment of obesity.
Throughout this specification and claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.